Systems and methods for extracting non-polar lipids from an aqueous algae slurry and lipids produced therefrom

ABSTRACT

Methods, systems, and apparatuses for extracting non-polar lipids from microalgae are achieved using a lipid extraction device having an anode and a cathode that forms a channel and defines a fluid flow path through which an aqueous slurry is passed. An electromotive force is applied across the channel at a gap distance in a range from 0 5 mm to 200 mm to cause the non-polar lipids to be released from the algae cells. The non-polar lipids can be extracted at a high throughput rate and with low concentrations of polar lipids such as phospholipids and chlorophyll.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/642,096 (Attorney Docket No. 18990.55), filed on Dec. 18, 2012,titled “Systems and Methods for Extracting Non-Polar Lipids from anAqueous Algae Slurry and Lipids Produced Therefrom”, which is anationalization of Patent Cooperation Treaty Application No.PCT/US2010/053260 (Attorney Docket No. 18990.8), filed on Oct. 19, 2010,titled “Systems and Methods for Extracting Non-Polar Lipids from anAqueous Algae Slurry and Lipids Produced Therefrom”, which claimspriority to U.S. patent application Ser. No. 12/907,024 (Attorney DocketNo. 18990.7), filed Oct. 18, 2010, titled “Systems, Apparatuses andMethods for Extracting Non-Polar Lipids from an Aqueous Algae Slurry andLipids Produced Therefrom”, which is a nationalization of PatentCooperation Treaty Application No. PCT/US2010/031756 (Attorney DocketNo. 18990.6), filed Apr. 20, 2010, titled “Systems, Apparatus andMethods for Obtaining Intracellular Products and Cellular Mass andDebris from Algae and Derivative Products and Process Use Thereof”,which claims priority to U.S. Provisional Patent Application No.61/170,698 (Attorney Docket No. 18990.5), filed Apr. 20, 2009, titled“Cell Lysing and Apparatus”. The disclosures of each of the applicationsto which the present application claims priority are incorporated byreference.

FIELD OF THE INVENTION

The invention relates to the fields of energy and microbiology. Inparticular, the invention relates to systems, apparatus and methods forharvesting cellular mass and debris as well as intracellular productsfrom algae cells which can be used as a substitute for fossil oilderivatives in various types of product manufacturing.

BACKGROUND

The intracellular products of microorganisms show promise as a partialor full substitute for fossil oil derivatives or other chemicals used inmanufacturing products such as pharmaceuticals, cosmetics, industrialproducts, biofuels, synthetic oils, animal feed, and fertilizers.However, for these substitutes to become viable, methods for obtainingand processing such intracellular products must be efficient andcost-effective in order to be competitive with the refining costsassociated with fossil oil derivatives. Current extraction methods usedfor harvesting intracellular products for use as fossil oil substitutesare laborious and yield low net energy gains, rendering them unviablefor today's alternative energy demands. Such methods can produce asignificant carbon footprint, exacerbating global warming and otherenvironmental issues. These methods, when further scaled up, produce aneven greater efficiency loss due to valuable intracellular componentdegradation and require greater energy or chemical inputs then what iscurrently financially feasible from a microorganism harvest. Forexample, the cost per gallon for microorganism bio-fuel is currentlyapproximately nine-fold over the cost of fossil fuel.

Recovery of intracellular particulate substances or products frommicroorganisms requires disruption or lysing of the cell transmembrane.All living cells, prokaryotic and eukaryotic, have a plasmatransmembrane that encloses their internal contents and serves as asemi-porous barrier to the outside environment. The transmembrane actsas a boundary, holding the cell constituents together, and keeps foreignsubstances from entering. According to the accepted current theory knownas the fluid mosaic model (S. J. Singer and G. Nicolson, 1972), theplasma membrane is composed of a double layer (bi-layer) of lipids, anoily or waxy substance found in all cells. Most of the lipids in thebilayer can be more precisely described as phospholipids, that is,lipids that feature a phosphate group at one end of each molecule.

Within the phospholipid bilayer of the plasma membrane, many diverse,useful proteins are embedded while other types of mineral proteinssimply adhere to the surfaces of the bilayer. Some of these proteins,primarily those that are at least partially exposed on the external sideof the membrane, have carbohydrates attached and therefore are referredto as glycoproteins. The positioning of the proteins along the internalplasma membrane is related in part to the organization of the filamentsthat comprise the cytoskeleton, which helps anchor them in place. Thisarrangement of proteins also involves the hydrophobic and hydrophilicregions of the cell.

Intracellular extraction methods can vary greatly depending on the typeof organism involved, their desired internal component(s), and theirpurity levels. However, once the cell has been fractured, these usefulcomponents are released and typically suspended within a liquid mediumwhich is used to house a living microorganism biomass, making harvestingthese useful substances difficult or energy-intensive.

In most current methods of harvesting intracellular products from algae,a dewatering process has to be implemented in order to separate andharvest useful components from a liquid medium or from biomass waste(cellular mass and debris). Current processes are inefficient due torequired time frames for liquid evaporation or energy inputs requiredfor drying out a liquid medium or chemical inputs needed for a substanceseparation.

Accordingly, there is a need for a simple and efficient procedure forharvesting intracellular products from microorganisms that can be usedas competitively-priced substitutes for fossil oils and fossil oilderivatives required for manufacturing of industrial products.

BRIEF SUMMARY

The present invention relates to methods, systems, and apparatuses forextracting non-polar lipids from microalgae and to the lipid productsextracted from these methods, systems and apparatuses. The methods,systems, and apparatuses of the invention can advantageously extract thenon-polar lipids from microalgae at a high volume flow rate. Byextracting the non-polar lipids (e.g., triglycerides) separate from thepolar lipids (e.g., phospholipids and chlorophyll) and cellular debris,the methods, systems, and apparatuses of the invention can produce aproduct suitable for use in traditional petrochemical processes such aspetrochemical processes that utilize precious metal catalysts.

In one embodiment, the present invention relates to a method forextracting non-polar lipids from microalgae in a flowing aqueous slurry.The method includes (i) providing an aqueous slurry includingmicroalgae; (ii) providing a lipid extraction apparatus having a bodyincluding a channel that defines a fluid flow path, at least a portionof the channel formed from a cathode and an anode spaced apart to form agap with a distance in a range from 1 mm to 200 mm within the channel;(iii) flowing the aqueous slurry through the channel and applying anelectromotive force across the gap, the electromotive force compromisingthe microalgae cells and releasing a lipid fraction having greater than80 wt % non-polar lipids and less than 20 wt % polar lipids; and (iv)recovering at least a portion of the nonpolar lipid fraction.

By selecting the gap distance, voltage, amperage and flow rate, themicroalgae can be lysed or otherwise compromised to release non-polarlipids without extracting the polar lipids such as the phospholipids andthe chlorophyll. Moreover, since the anode and the cathode form part ofthe channel through which the aqueous slurry is flowing, the microalgaecan be exposed to a large surface area of anode and cathode atreasonable distances, which improves the efficiency and economy of lipidextraction and allows high throughput and scalability.

In addition, since the anode and cathode form part of a channel, theduration of the algae in the field can be controlled by adjusting theflow rate in the channel (e.g., by adjusting the pumping pressure). Theability to adjusting the flow rate, amperage, and/or voltage is usefulfor processing microalgae because some properties of microalgae slurriescan vary over time due to naturally occurring variations. Thus, themethods systems and apparatuses of the invention allow extraction thataccommodates these variations.

The present invention is also directed to the lipid fraction producedfrom the methods, systems, and apparatuses described herein. The lipidfraction released from the microalgae cells using the methods, systems,and apparatuses of the present invention can have a unique compositiondue to the way in which the lipids are released. The process can becarried out by controlling the gap distance, voltage, amperage, and flowrate to release the vast majority of non-polar lipids without releasingthe polar lipids. The particular voltages, amperages, and flow rateswill depend on the particular aqueous slurry and species of microalgaebeing process. However, a visual inspection of the released lipidfraction can indicate when the polar lipids fraction is being extractedin large quantities since the undesired polar lipids (e.g., mixtures ofchlorophyll and phospholipids) tend to be darker. Alternatively, theprocess can include sampling the released lipid fraction and analyzingthe sample using high performance liquid chromatography to determine thepercentage of undesired polar lipids. The flow rate, amperage, voltage,and/or gap distance can then be selected to minimize the percentage ofpolar lipids while maintaining suitable throughput. In one embodiment, acomputer controlled lipid extraction apparatus can use HPLC data toselect the parameters that minimize polar lipids in the released lipidfraction. In a preferred embodiment, the non-polar lipid content in thereleased fraction is greater than 90% and the polar fraction is lessthan 10%, more preferably the non-polar lipid content is greater than95% and the polar lipid content is less than 5%, even more preferablythe non-polar lipid content is greater than 98% and the polar lipidcontent is less than 2%, and most preferably the non-polar lipid contentis at least 99% and the polar lipid content is less than 1%.

The composition of the released lipid fraction will also depend to somedegree on the aqueous slurry used for the feed. In one embodiment of theinvention, the released lipid fraction is recovered from a process usingan aqueous slurry where at least 70 wt % of the microorganisms in theslurry are microalgae (preferably at least 80 wt %, more preferably atleast 90 wt %, and most preferably at least 99 wt % microalgae).

The present invention is also directed to lipid extraction apparatusesand systems. In one embodiment, the lipid extraction apparatus includesa body including a channel that defines a fluid flow path from a firstopening to a second opening, the first opening providing an inlet for anaqueous algae slurry and the second opening providing an outlet for theaqueous algae slurry; a cathode, an anode, and an insulator forming atleast a portion of the channel that defines the fluid flow path, thecathode and the anode being spaced apart to form a gap with a distancein a range from 1 mm to 200 mm. The anode and the cathode providesufficient surface area at the gap distance such that the volume of thefluid flow path within the gap is at least 50 ml, preferably at least100 ml, and most preferably at least 200 ml. The narrow gap distance andlarge volume of fluid flow can be achieved by either making the channellong or wide or both. However, by limiting the gap distance, theapparatus can apply an electromotive force suitable for extractingnon-polar lipids, while allowing high throughput.

In one embodiment, the channel of the lipid extraction apparatus can beformed from first and second electrically conductive tubes that areconfigured to be a tube within a tube, where the spacing between theinner and outer tube forms the fluid flow path and the inner and outerelectrically conductive tubes provide the cathode and anode of theapparatus. In this embodiment, an insulator can be placed between thefirst and second electrically conductive tubes to prevent a short acrossthe tubes and to optionally direct fluid flow. In one embodiment, theapparatus includes rifling between the first and second tubes to cause aspiral flow path. This can be accomplished using a spacer, grooves,protrusions, or other suitable structure that can cause directionalfluid flow between the two electrically conductive tubes.

These and other objects and features of the present invention willbecome more fully apparent from the following description and appendedclaims, or may be learned by the practice of the invention as set forthhereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to describe the manner in which the above-recited and otheradvantages and features of the invention can be obtained, a moreparticular description of the invention briefly described above will berendered by reference to specific embodiments thereof which areillustrated in the appended drawings. Understanding that these drawingsdepict only typical embodiments of the invention and are not thereforeto be considered to be limiting of its scope, the invention will bedescribed and explained with additional specificity and detail throughthe use of the accompanying drawings in which:

FIG. 1 illustrates a portion of a lipid extraction device according toone embodiment of the invention;

FIG. 2 illustrates a sectional perspective view of biomass flowing inbetween the anode and cathode wall surfaces of the device of FIG. 1;

FIG. 3 illustrates a lipid extraction apparatus with a flowing liquidmedium containing a microorganism biomass being exposed to anelectromagnetic field caused by an electrical transfer;

FIG. 4 illustrates an overview of a normal sized microorganism cell inrelationship to a secondary illustration of a swollen cell duringexposure to an electromagnetic field and electrical charge;

FIG. 5 illustrates the lipid extraction apparatus of FIG. 4 with heatbeing applied and transferred into the liquid medium;

FIG. 6 illustrates a perspective view of the anode and cathode tubes ofan apparatus according to one embodiment of the invention;

FIG. 7 illustrates a perspective sectional view of the apparatus of FIG.6 and including a spiral spacer in between the anode and cathode tubes;

FIG. 8 is a perspective view of a series of lipid extraction devices ofFIG. 7 connected in parallel by an upper and lower manifold;

FIG. 9 depicts a general flow diagram illustrating various steps of aprocess for extracting non-polar lipids from microalgae according to oneembodiment of the present invention;

FIG. 10 depicts a general flow diagram illustrating various steps of aprocess for extracting non-polar lipids from microalgae according to oneembodiment of the present invention;

FIG. 11 illustrates a side view of a micron mixer in association with asecondary tank containing a biomass and sequences of developing foamlayers generated by a micron mixer;

FIG. 12 illustrates a secondary tank containing the liquid medium and aresulting foam layer capable of being skimmed off the surface of theliquid medium, into a foam harvest tank;

FIG. 13 illustrates one embodiment of a method and apparatus (system) asdescribed herein for the harvest of useful substances from an algaebiomass involving extraction with an emf;

FIG. 14 illustrates another embodiment of a method and apparatus(system) as described herein for the harvest of useful substances froman algae biomass using a lipid extraction device that applies a pulsedemf (i.e. EMP);

FIG. 15 illustrates an example of a modified static mixer; and

FIG. 16 is a table of data from experiments to quantify lipid extractionand identify optimal extraction parameters.

DETAILED DESCRIPTION

The present invention relates to methods, systems, and apparatuses forextracting non-polar lipids from microalgae and to the lipid productsextracted from these methods, systems and apparatuses. The methods,systems, and apparatuses of the invention can advantageously extract thenon-polar lipids from microalgae at a high volume flow rate. Byextracting the non-polar lipids (e.g., triglycerides) separate from thepolar lipids (e.g., phospholipids and chlorophyll) and cellular debris,the methods, systems, and apparatuses of the invention can produce aproduct suitable for use in traditional petrochemical processes such aspetrochemical processes that utilize precious metal catalysts.

In a first embodiment, a method is described for extracting non-polarlipids from microalgae. The method generally includes (i) providing anaqueous slurry including microalgae; (ii) providing a lipid extractionapparatus having a body including a channel that defines a fluid flowpath, at least a portion of the channel formed from a cathode and ananode are spaced apart to form a gap with a distance in a range from 1mm to 200 mm within the channel; (iii) flowing the aqueous slurrythrough the channel and applying an electromotive force across the gap,the electromotive force compromising the microalgae cells and releasinga lipid fraction having greater than 80 wt % non-polar lipids and lessthan 20 wt % polar lipids; and (iv) recovering at least a portion of thenon-polar lipid fraction.

In performing the method, a microalgae slurry is provided. Themicroalgae slurry includes water and algae. Because the process of theinvention is carried out using an aqueous slurry, the costs normallyassociated with drying the algae before extraction can be avoided. Thealgae cells can be any microalgae cells, including, but not limited to,Nanochloropsis oculata, Scenedesmus, Chlamydomonas, Chlorella,Spirogyra, Euglena, Prymnesium, Porphyridium, Synechoccus sp,Cyanobacteria and certain classes of Rhodophyta single celled strains.The algae can be phototrophic algae grown in an open natural environmentor in a closed environment. The methods of the invention can also beused to extract lipids from heterotrophic bacterial.

The concentration of the algae in the slurry will depend in part on thetype of algae, the growth conditions, and whether the algae has beenconcentrated. The aqueous slurry can include be grown and used at anysuitable concentration, such as, but not limited to a range from about100 mg/L to about 5 g/l (e.g., about 500 mg/L to about 1 g/L). In someembodiments, unconcentrated algae from a growth vessel will be from 250mg/L to 1.5 g/L and may be pre-concentrated with other conventionalmeans to within a range from 5 g/L to 20 g/L. If desired, the microalgaeconcentration can be increased using any known technique. For example,concentrating can be carried out using flocculation. The flocculationcan be a chemical flocculation or electro-flocculation or any otherprocess that effectuates a similar function.

In one embodiment, the algae slurry has a desired concentration ofmicroalgae as a percentage of the total microorganisms in the slurry.The purity of the slurry with respect to the concentration of microalgaecan impact the composition of the lipids released from the extractionprocess. In a preferred embodiment, at least 70 wt % of microorganismwithin the aqueous slurry are microalgae, preferably at least 80 wt %,more preferably at least 90 wt %, even more preferably at least 95 wt %,and most preferably at least 99 wt %.

The pH of the slurry during extraction can vary. However, in oneembodiment, the pH is alkaline. Acid or base can be added to keep the pHcan be kept in a range from 6.69.0, 6, 8-8.6, or 7.0-8.5.

In a second step, a lipid extraction apparatus is provided that includesan anode and a cathode that form a channel through which the aqueousslurry can flow. FIG. 1 is a schematic of a portion of a lipidextraction device 100 that is suitable for use in the method of theinvention. The portion of extraction device 100 includes a body 102 thathas an anode 104 and a cathode 106 electrically separated by aninsulator 108. Anode 104 and cathode 106 are spaced apart to form achannel 112 that defines a fluid flow path 110. Channel 112 has a length116 that extends the length of the anode and cathode exposed to thefluid flow path 110. Channel 112 also has a width 118 that is defined bythe space between the insulators that is exposed to the anode 104 andcathode 106. The gap 114 between anode 104 and cathode 106 has adistance suitable for applying an emf through the aqueous algae slurry.In one embodiment, gap 114 is in a range from 0 5 mm to 200 mm,preferably 1 mm to 50 mm, more preferably 2 mm 20 mm. The narrow gapdistance coupled with a large width 118 and length 116 can provide alarge volume for channel 112 while maintaining a strong electrical fieldfor compromising the algae cells to release polar lipids. The length 116of channel 112 is the dimension in the direction of fluid flow 110 andcan be any length so long as channel is not hampered by plugging orsignificant pressure drops. In one embodiment, the length 116 of channel112 is at least 25 cm, preferably 50 cm, more preferably 100 cm, andmost preferably at least 200 cm. In one embodiment the length 116 can beless than 1000 cm, less than 500 cm or less than 250 cm. The width canbe any width so long as the materials of the anode and cathode aresufficiently strong to span the width without contacting one another. Ina preferred embodiment, the volume of the channel between the anode andcathode and within the gap distance 114, (i.e., the gap volume) is atleast 50 ml, more preferably at least 200 ml, even more preferably atleast 500 ml, and most preferably at least 1 liter. In one embodiment,the surface area of the anode and the cathode exposed to the fluid flowis at least 500 cm², preferably at least 1000 cm², and more preferablyat least 2000 cm².

The anode 104 and cathode 106 can be made of any electrically conductivematerial suitable for applying emf across the gap, including but notlimited to metals such as steel and conductive composites or polymers.

The shape of the anode and cathode can be planer or cylindrical or othershape. As described more fully below, an annulus created between aninner metallic surface of a tube and an outer surface of a smallermetallic conductor tube placed within the large tube is preferred forits ability to avoid fouling and to maintain a high surface area in acompact design. The tubes need not have a circular periphery as an inneror outer tube may be square, rectangular, or other shape and the tubeshape does not necessarily have to be the same, thereby permitting tubeshapes of the inner and outer tubes to be different. In a most preferredembodiment, the inner conductor and outer tube are concentric tubes,with at least one tube, preferably the outer tube, being provided with aplurality of spiral grooves separated by lands to impart a rifling tothe tube. This rifling has been found to decrease build-up of residue onthe tube surfaces. In commercial production, there may be a plurality ofinner tubes surrounded by an outer tube to increase the surface contactof the metal conductors with the algae.

Furthermore, the use of electrical insulators, such as plastic tubes,baffles, and other devices, can be used to separate a large lipidextraction devices into a plurality of zones, so as to efficientlyscale-up the invention to commercial applications.

In performing the method, the aqueous algae slurry is fed through thechannel along the fluid flow path between the anode and cathode (i.e.,through the gap). Power is applied to the anode and cathode to producean electromotive force that compromises or lyses the algae cells torelease the non-polar lipids (also referred to herein as “single stepextraction” or “SSE”). For a given gap distance or channel volumebetween the anode and cathode, the amperage, flow rate, and voltage areselected to effectuate the release of the non-polar lipids.

Referring to FIG. 2, apparatus 100 is shown in cross section with anaqueous algae slurry 120 disposed between cathode 106 and anode 104. Theaqueous algae slurry 120 is caused to flow through channel 112 using apump (not shown). By way of an electrical conduit, a negative connection122 is made to the anode 104, which provides electrical grounding.Positive electrical input 124 also delivered by way of a conduitconnection provide positive electrical transfer throughout the cathode106. When a positive current 124 is applied to the cathode 106 it thenseeks a grounding circuit for electrical transfer as indicated by arrow126 or in this case, to the anode 104, which allows the completion ofthe electrical circuit. In this respect, transfer of electrons occursbetween the positive and negative surfaces areas but only when anelectrically conductive liquid is present between them. As the liquidmedium containing the algae slurry 120 is flowed between the surfaceareas, electrical transfer from the cathode 106 through the slurry 120to the anode 104 is made. As a liquid containing a microorganism biomasstransverses the anode and cathode circuit, the cells are exposed to theelectric field that causes expansion and contraction of the cells.

In reference to FIG. 3, a simplified schematic is used to illustrate anemf transfer between two electrical conductive metal pieces with aliquid medium containing a living microorganism biomass flowing betweenthem in a method for harvesting biomass from an aqueous solutioncontaining algae cells. The cathode 106 requires a positive electricalconnection point 128, which is used for positive current input. Positivetransfer polarizes the entire length and width of the cathode 106 andseeks a grounding source in anode 104. In order to complete anelectrical circuit, the anode 104 includes a grounding connection point127, which allows an electrical transfer 132 to occur through aqueousslurry 120. The aqueous slurry includes a liquid medium that contains anutrient source mainly composed of a conductive mineral content that wasused during a growth phase of the algae in aqueous slurry 120. Theliquid medium containing the nutrient source further allows positiveelectrical input to transfer between the cathodes 106 through the liquidmedium/biomass 120 to the anode 104 and which only occurs when theliquid medium is present or flowing. Electrical input causes cellularelongation such as the distention shown in algae 130 b as compared toalgae 130 a.

In reference to FIG. 4, a simplified illustration is used to exhibit thedifference between a normal sized microalgae cell 130 a in comparison toa microalgae cell 130 b, which has been extended by the electrical fieldbetween the cathode and anode. During the electrical “on” phase, emf 132(FIG. 3) polarizes the algae cell walls and/or membranes. A positivecharge and a negative charge develop on the membrane of respective ends134 and 135 of algae cell 130 b in alignment with the emf field 132. Thedipole on the cells causes the cells to be pulled apart along theelectrical field lines until the cell wall/membrane is compromised,thereby releasing the cell contents. This elongation eventually causesexternal structural damage to the exterior wall with general damageresulting in a wall and membrane that is permeable to the intracellularfluids and/or causes lysis. The flow rate, voltage, and amperage, areselected in combination with the gap distance and composition of theaqueous slurry to cause release of primarily the polar lipids withoutreleasing the non-polar lipids such as those in the cell membrane andthe chlorophyll. A visual inspection or high performance liquidchromatography can be used to monitor the lipid content to minimize thepolar lipid fraction as compared to the non-polar lipid fraction.

In one the flow rate through the gap volume (i.e., the portion of thechannel in the electric field at the gap distance) is 0.1 ml/second perml of gap volume, more preferably at least 0.5 ml/second per ml of gapvolume, even more preferably at least 1.0 ml/second per ml of gap volumeand most preferably at least 1.5 ml/second per ml of gap volume. In oneembodiment the flow rate can be controlled controlling the pressureusing a pump or other suitable fluid flow mechanical devices.

The average amperage can be at least 1 amp, 5 amps, 10 amps, 50 amps, oreven at least 100 amps. The maximum amps can be less than 200 amps, lessthan 100 amps, less than 50 amps, or less than 10 amps. The range ofamperage can be any range from the foregoing maximum and minimumamperages.

The voltage can be at least 1V, 10V, 100V, 1 kV, or even at least 20 kV.The maximum voltage can be less than 50 kV, less than 30 kV, less than10 kV, less than 1 kV, or less than 100V. The range of voltage can beany range of the foregoing maximum and minimum voltages.

An example of a suitable configuration for extracting non-polar lipidsincludes an apparatus with a gap distance of 1/16-¼ inch and a gapvolume of 250 ml-1000 ml and an electrical current of 1-60 peak amps @1-24 volts or 25 w to 500 watts. The flow volume can be at a rate of 1gallon per minute (GPM) of throughput with a culture having a density of500 mg/L, one would use approximately 70 watts of energy (3.5v @ 20 peakamps) for a successful extraction. At 5 GPM, the same culture could beextracted using approximately 350 watts (3.5v @ 100 peak amps).

In another example, at 0.5 GPM, 500 mg/L density, an electrical currentof approximately 60 watts (15 peak amps @ 4 volts) is applied.Generally, a GPM of approximately 0.1 to approximately 5 GPM and wattsin the range of about 20 to about 1000 watts (e.g., 2-18 volts @ 2-50peak amps) are used. For example, at 1 GPM of throughput with a culturehaving a density of 500 mg/L, one could use approximately 70 watts ofenergy (3.5v @ 20 peak amps) for a successful extraction. At 5 GPM, thesame culture would require approximately 350 watts (3.5v @ 100 peakamps).

In one embodiment, the emf can be pulsed on and off repeatedly to causeextension and relaxation of the algae cells. In this embodiment,voltages can be higher and peak amperage lower while average amperageremains relatively low. This reduces the energy requirements foroperating the apparatus and reduces wear on the anode and cathode. Inone embodiment, the frequency of the emf pulses is at least about 500Hz, 1 kHz, 2 kHz, or 30 kHz. The frequency can be less than 200 kHz, 80,kHz, 50 kHz, 30 kHz, 5 kHz, or 2 kHz. Ranges for the pulse frequency canbe any combination of the foregoing maximum and minimum frequencies.

The temperature of the aqueous slurry during extraction can also have animpact on the power required to extract the non-polar lipids. Lipidextraction may be carried out at room temperature. However, in oneembodiment, heat is added to the aqueous algae slurry to achieve adesired temperature. Lipid extraction may be carried out at atemperature above 40° F., 65° F., 80° F., 100° F., or 120° F. Thetemperature can be below 130° F., 115° F., 105° F., or 90° F. Ranges forthe extraction temperature can be any combination of the foregoingmaximum and minimum temperatures.

The temperature of the slurry can also be adjusted to control thespecific gravity of the water relative to the algae (the specificgravity of water density is optimal at 40 degrees F.). As the liquidmedium (typically mainly composed of water) is heated, alterations toits hydrogen density occurs; this alteration of density allows anormally less dense material to sink or in this case, heavier fracturedcellular mass and debris materials which would normally float, nowrapidly sink to the bottom of the liquid column. This alteration alsoallows easier harvesting of these materials which are also useful forother product applications. Once the EMP and heating process has beenachieved, the liquid medium containing a now fractured biomass istransferred into a secondary holding tank where a liquid pump allows acontinuous loop flow. As used in this description “specific gravity” isa dimensionless unit defined as the ratio of density to a specificmaterial as opposed to the density of the water at a specifiedtemperature.

In reference to FIG. 5, a simplified schematic is used to illustrate aheat transfer example between the outer walls of the cathode 106 and/oranode 104 and into the liquid medium/biomass during the EMP process in amethod for harvesting cellular mass and debris from an aqueous solutioncontaining algae cell. An applied heating device 134 attaches to theoutside wall surfaces of the cathode 106 and anode 104, which allowsheat transfer to penetrate into the aqueous slurry 120.

The products recovered from the methods of the present invention canhave a relatively low content of polar lipids such as chlorophyll andphospholipids. In a preferred embodiment the lipid extraction accordingto the present invention is carried out to produce a released lipidfraction with a non-polar lipid content greater than 90% and the polarfraction is less than 20%, preferably the non-polar lipid content in thereleased fraction is greater than 90% and the polar content is less than10%, even more preferably the non-polar lipid content is greater than95% and the polar lipid content is less than 5%, yet even morepreferably the non-polar lipid content is greater than 98% and the polarlipid content is less than 2%, and most preferably the non-polar lipidcontent is at least 99% and the polar lipid content is less than 1%.

The methods of the invention may further include reducing the content ofphosphorus to less than 100 ppm, preferably less than 20 ppm and mostpreferably less than 10 ppm and using the non-polar lipids in at leastone catalytic refining process. For example, the lipids can behydrotreated using a supported catalyst.

In one embodiment, the method of extracting lipids can be carried out byperiodically drawing algae from a growing algae source to maintain asteady rate of growth. Steady state growth can be achieved by drawingalgae at a rate of less than half the algae mass per unit time that ittakes for the algae to double. In one embodiment algae is harvest atleast as often as the doubling time of the algae, more preferably atleast twice during the doubling time of the algae. The doubling timewill depend on the algae type and growth conditions but can be as littleas 6 hours to several days.

FIGS. 6-8 describe an example lipid extraction apparatus in more detail.The apparatus 222 shown in FIGS. 6-8 illustrate a “tube within a tube”configuration. FIG. 6 illustrates a disassembled lipid extraction deviceshowing a first conductive tube 203 (hereinafter cathode 203, althoughconductive tube 203 may also be the anode or switch between anode andcathode) that is configured to be placed in a second conductive tube 202(hereinafter anode 202, although conductive tube 202 may also be thecathode or switch between anode and cathode). The outer anode tube 202includes a pair of containment sealing end caps 207 and 208. Sealing endcap 207 provides an entry point 209 used to accept an aqueous algaeslurry. After biomass transiting, the opposing end cap 208 provides anexit point 210 to the outward flowing algae biomass.

As shown also in FIG. 6, the inner cathode tube 203 includes sealed endcaps 211 and 212 to prevent liquid flow through the center of the tubeand to divert the flow between the inner surface of anode 202 and theouter surface of cathode 203, thereby forming a channel. The channel canbe sized and configured as described above with respect to FIG. 1. Theuse of a “tube within a tube” configuration is particularly advantageousfor avoiding fouling by the algae and/or other organism in the slurry.

FIG. 7 shows an alternative embodiment in which an insulative spacer 213that is positioned in the channel between the anode and cathode to causespiraling fluid flow. Insulative spiraling isolator spacer 213 serves asa liquid seal between the two wall surfaces 214 and 215 with thethickness of the spacer preferably providing equal distance spacingbetween the anode 202 and the cathode 203. The spacing and directionalflow can cause the fluid flow path to complete three hundred and sixtydegree transfer of electrical current around the anode 202 and cathodetube 203. The spacer 213 can also help prevent contact between the anode202 and cathode 203, which prevents shorting the anode and the cathodeand forces electrical current through the liquid medium. Further thespiraling isolator 213 now provides a gap 216 between the two wallsurfaces 214 and 215 allowing a passage way for a flowing biomass 201.The spiraling directional flow further provides a longer transitduration which provides greater electrical exposure to the flowingbiomass 1 thus increasing substance extraction efficiency at a lower perkilowatt hour consumption rate during intracellular substanceextraction. Any suitable material can be used as a spacer. Typically,ceramic, polymeric, vinyl, PVC plastics, bio-plastics, vinyl,monofilament, vinyl rubber, synthetic rubber, or other non-conductivematerials are used.

In reference to FIG. 8, a series of anode and cathode circuits 222 areshown in parallel having a common upper manifold chamber 218 whichreceives an in flowing biomass 1 through entry port 20. Once enteringinto the upper manifold chamber 218, the biomass 1 makes a downwardconnection into each individual anode and cathode circuit 222 throughentry ports 209 which allow a flowing connection to the sealing end caps208. It is at this point where the flowing biomass 201 (i.e., aqueousalgae slurry) enters into the anode and cathode circuits 222. Oncetransiting in spiral through the individual circuits 222, the flowingbiomass 201 exits into a lower manifold chamber 219 where the biomass201 is then directed to flow out of the apparatus 200 (system) throughexit point 221.

With Reference to FIG. 9, an overall process is described for extractingand processing lipids. The methods, systems, and apparatuses of theinvention can use all or a portion of the steps and apparatuses shown inFIG. 1. In a method of harvesting at least one intracellular productfrom algae cells in aqueous suspension, the cells are grown in a growthchamber. A growth chamber (also referred to herein as a “reactor”) canbe any body of water or container or vessel in which all requirementsfor sustaining life of the algae cells are provided for. Examples ofgrowth chambers include an open pond or an enclosed growth tank. Thegrowth chamber is operably connected to an apparatus 200 as describedherein such that algae cells within the growth chamber can betransferred to the apparatus 200, e.g., by way of gravity or a liquidpump, the living bio mass is flowed via a conduit into the inlet sectionof the anode and cathode circuit. Algae cells within the growth chambercan be transferred to the apparatus 200 by any suitable device orapparatus, e.g., pipes, canals, or other conventional water movingapparatus. In order to harvest at least one intracellular product fromthe algae cells, the algae cells are moved from the growth chamber to anapparatus 200 (or other apparatus as described above with reference toFIGS. 1-8) and contained within the apparatus 200. When added to theapparatus 200, the algae cells are generally in the form of a liveslurry (also referred to herein as “biomass”). The live slurry is anaqueous suspension that includes algae cells, water and nutrients suchas an algal culture formula based on Guillard's 1975 F/2 algae foodformula that provides nitrogen, vitamins and essential trace mineralsfor improved growth rates in freshwater and marine algae. Any suitableconcentration of algae cells and sodium chloride, fresh, brackish orwaste water can be used, such that the algae cells grow in the aqueoussuspension.

After the non-polar lipid fraction is released in apparatus 200, thereleased lipid fraction may be subjected to one or more downstreamtreatments including gravity clarification. Gravity clarificationgenerally occurs in a clarification tank in which the intracellularproduct(s) of interest (e.g., lipids) rises to the top of the tank, andthe cellular mass and debris sinks to the bottom of the tank. In such anembodiment, upon transiting the circuit, the fractured cellular mass anddebris is flowed over into a gravity clarification tank that is operablyconnected to an apparatus 200 for harvesting cellular mass and debrisand intracellular products from algae cells as described herein. In thegravity clarification tank, the lighter, less dense substances float tothe top of the liquid column while the heavier, denser remains sink tothe bottom for additional substance harvest.

The intracellular product(s) of interest is then easily harvested fromthe top of the tank such as by skimming or passing over a weir, and thecellular mass and debris can be discarded, recovered and/or furtherprocessed. A skimming device then can be used to harvest the lightersubstances floating on the surface of the liquid column while theheavier cellular mass and debris remains can be harvested from thebottom of the clarification tank. The remaining liquid (e.g., water) canbe filtered and returned to the growth chamber (recycled) or removedfrom the system (discarded).

In an embodiment in which the intracellular product is oil (i.e.,lipids), the oil can be processed into a wide range of productsincluding vegetable oil, refined fuels (e.g., gasoline, diesel, jetfuel, heating oil), specialty chemicals, nutraceuticals, andpharmaceuticals, or biodiesel by the addition of alcohol. Intracellularproducts of interest can be harvested at any appropriate time,including, for example, daily (batch harvesting). In another example,intracellular products are harvested continuously (e.g., a slow,constant harvest). The cellular mass and debris can also be processedinto a wide range of products, including biogas (e.g., methane,synthetic gas), liquid fuels (jet fuel, diesel), alcohols (e.g.,ethanol, methanol), food, animal feed, and fertilizer.

In addition to gravity clarification, any suitable downstream treatmentcan be used. Possible downstream treatments are numerous and may beemployed depending on the desired output/use of the intracellularcontents and/or bio cellular mass and debris mass. For example, lipidscan be filtered by mechanical filters, centrifuge, or other separationdevice, for example, then heated to evacuate more water. The lipids canthen be further subjected to a hexane distillation. In another example,cellular mass and debris can be subjected to an anaerobic digester, asteam dryer, or belt press for additional drying for food, fertilizeretc. As shown in FIG. 9, downstream treatments also include, e.g.,polishing and gravity thickening.

In one embodiment, the present invention includes a method of harvestingcellular mass and debris from an aqueous solution containing algae cellsby subjecting algae cells to pulsed emf and to cavitation (i.e.,microbubbles) in an apparatus as described herein, resulting in amixture that includes both intracellular product(s) (e.g., lipids) andcellular mass and debris. A process flow diagram that includes acavitation step is shown in FIG. 10. The methods and apparatus of thisembodiment can use any of the lipid extraction devices described herein.The cells can be subjected to cavitation before application of (upstreamof) pulsed emf (i.e., “EMP”), or they may be subjected to cavitationconcomitantly with EMP (see FIG. 15 that depicts the cavitation deviceelectrified as it would be the EMP conductor). In one embodiment, acavitation device includes an anode, cathode and venture mixer (all inone). In this embodiment, the cavitation unit is reduced (e.g., byhalf), a non-conductive gasket is added, and it is electrified. Undernormal pressure conditions, e.g., under 100 psi, no effect was observedwhen cavitation was applied upstream of EMP, however, at pressures above100 psi (e.g., 110, 115, 120, 130, 140, 150, 200, 300, 400 psi, etc.),it may have an effect.

In the method where cavitation is used, a micron mixing device, such asa static mixer or other suitable device such as a high throughputstirrer, blade mixer or other mixing device is used to produce a foamlayer composed of microbubbles within a liquid medium containing apreviously lysed microorganism biomass. Any device suitable forgenerating microbubbles, however, can be used. Following micronization,the homogenized mixture begins to rise and float upwards. As thismixture passes upwards through the liquid column, the less densevaluable intracellular substances freely attach to the rising bubbles,or due to bubble collision, into a heavier sinking cellular mass anddebris waste, (now allowed to sink due to heated water specifics). Therising bubbles also shake loose trapped valued substances (e.g., lipids)which also freely adhere to the rising bubble column. Once the foamlayer containing these useful substances has risen to the top of theliquid column, they now can be easily skimmed from the surface of theliquid medium and deposited into a harvest tank for later productrefinement. Once the foam layer rises to the top of the secondary tank,the water content trapped within the foam layer generally results inless than 10% (e.g., 5, 6, 7, 8, 9, 10, 10.5, 11%) of the originalliquid mass. Trapped within the foam are the less dense usefulsubstances, and the foam is easily floated or skimmed off the surface ofthe liquid medium. This process requires only dewatering of the foam,rather than evaporating the total liquid volume needed for conventionalharvest purposes. This drastically reduces the dewatering process,energy or any chemical inputs while increasing harvest yield andefficiency as well as purity. In this method, water can be recycled tothe growth chamber or removed from the system.

Cellular mass and debris can be harvested at any appropriate time,including, for example, daily (batch harvesting). In another example,cellular mass and debris is harvested continuously (e.g., a slow,constant harvest).

Once the liquid medium has achieved passage through the EMP apparatus,it is allowed to flow over into a secondary tank (or directly into adevice that is located near the bottom of the tank). In this method ofdewatering, the secondary tank is a tank containing a micron bubbledevice or having a micron bubble device attached for desiredintracellular component separation and dewatering. After transmembranelysis, a static mixer or other suitable device (e.g., any static mixeror device which accomplishes a similar effect producing micro-bubbles)is used and is located at the lowest point within a secondary tank. Whenactivated, the static mixer produces a series of micron bubblesresulting in a foam layer to develop within the liquid medium. As theliquid medium is continuously pumped through the micro mixer, bubbledfoam layers radiate outwards through the liquid and begin to rise andfloat upwards. The less dense desired intracellular components suspendedwithin the liquid medium attach to the micron bubbles floating upwardsand flocculate to the surface or are detached from heavier sinkingbiomass waste, (allowed to sink due to specific gravity alterations) dueto rising bubble collision within the water column.

In this embodiment, FIG. 11 illustrates a lower mounting location for amicron mixer 327 when in association with secondary tank 328 andcontaining a previously fractured biomass 329 suspended within a liquidmedium. This liquid medium is then allowed to flow through a lowersecondary tank outlet 330 where it is directed to flow through conduit331 having a directional flow relationship with a liquid pump 332. Dueto pumping action, the liquid is allowed a single pass through, or tore-circulate through the micron mixer via a micron mixer inlet opening333. As liquid continues to flow through the micron mixer 327,microscopic bubbles 334 are produced which radiate outwards within theliquid column 335, forming a foam layer 336. As the process continues,the composed layer starts to rise upwards towards the surface of theliquid column 335. Once the foam layer 336 starts its upward journeytowards the surface of the liquid column 335, the pump 332 is shut down,and thus the micronization process is complete. This allows all micronbubbles 334 produced at the lower exit point of the micron mixer 327 torise to the surface and as they do, they start collecting valuableintracellular substances released into the liquid medium during the EMPprocess. This upward motion of the micron bubbles 334 also rubs or bumpsinto heavier downward-sinking cellular mass and debris, further allowingthe release of trapped lighter valuable substances that have bonded withheavier-sinking cellular mass and debris remains. Once detached, thesesubstances adhere to the micron bubbles 334 floating upwards towards thesurface.

In reference to FIG. 10, a simple illustration is used to show a methodfor harvesting a foam layer 436 containing approximately ten percent ofthe original liquid medium mass/biomass 401. As the foam layer 436containing the valuable intracellular internal substances rises to thesurface of the liquid medium 435, a skimming device 437 can be used toremove the foam layer 436 from the surface 438 of liquid medium 435. Theskimming device 437 located at the surface area of the secondary tank428 allows the foam layer 436 to be pushed over the side wall of thesecondary tank 428 and into a harvesting container 439 where the foamlayer 436 is allowed to accumulate for further substance harvestingprocedures.

FIG. 11 illustrates one embodiment of a method and apparatus (system) asdescribed herein for the harvest of useful substances from an algaebiomass. Microorganism algae are grown in a containment system 540 andat the end of an appropriate growth cycle are transferred into thesubstance recovery process. The algae biomass are flowed through anoptional micron bubble cavitation step 541, used to soften the outercellular wall structure prior to other bio substance recovery processes.

After the cavitation step 541 an optional heat process 542 can beapplied to change the gravity specifics of the liquid feed stock watercontaining the biomass. The heat option 542 allows a faster transfer ofparticular substances released during the harvest process. After thebiomass has reached an appropriate heat range, it is then allowed toflow through an electromagnetic pulse field, the EMP station 543 wheretransiting biomass cells are exposed to the electromagnetic transfersresulting in the fracturing of the outer cellular wall structures.

Once flowed through the EMP process 543, the fractured biomasstransitions into a gravity clarifier tank 544 where heavier matter(ruptured cell debris/mass) 545 sinks down through the water column asthe lighter matter (intracellular products) 546 rises to the surfacewhere it allows an easier harvest. The heavier sinking mass 545 gathersat the bottom of the clarifier tank 544 where it can be easily harvestedfor other useful substances. After substance separation and recovery,the remainder of the water column 547 is sent through a water reclaimingprocess and after processing is returned back into the growthcontainment system 540.

FIG. 12 illustrates another embodiment of a method and apparatus(system) as described herein for the harvest of useful substances froman algae biomass. Microorganism algae are grown in a containment system648 and at the end of an appropriate growth cycle are then transferredinto the substance recovery process. The substance recovery consists ofthe algae biomass being transferred into an optional heat process 649where the biomass water column is subjected to heat prior to the EMPstation 650. After the EMP process, the fractured biomass is thentransferred over into a cavitation station 651 where micron bubbles areintroduced at a low point in a water column containment tank 652. As themicro-bubbles rise through the water column, the valuable released biosubstances (intracellular products) 653 attach to the rising bubbleswhich float to the surface of the water column allowing an easier andfaster skimming process for substance recovery. After substancerecovery, the remainder of the water column is sent through a waterreclaiming process 654 and after processing is returned back into thegrowth system 648.

EXAMPLES

The present invention is further illustrated by the following specificexamples. In the experiments described below, Nanochloropsis oculatacells were used. The examples are provided for illustration only andshould not be construed as limiting the scope of the invention in anyway.

Example 1 Cell Lysing Method and Apparatus

In view of the interest in algae as a source of fuels and othermaterials, the development of methods and apparatuses for processingalgal cells on a large scale is of great utility in processing the algalcells for such purposes. Such methods and apparatuses are describedbelow.

One embodiment of a method for processing algal cells in suspensioninvolves passing algal cells in aqueous suspension through a staticmixer, where the static mixer creates cavitation effects, electrolyzingthe suspension, and separating lysed cells from water in the suspension.

In particular embodiments, the method also involves entraining a pH orORP modifying agent in the suspension, e.g., carbon dioxide. In such anembodiment, carbon dioxide typically is entrained in a static mixer. Ina further refinement, because alkaline materials may assist (make theprocess more efficient), agents may be used.

In certain embodiments, the method also involves collecting hydrogen gasgenerated by the electrolysis, e.g., at the mixer.

In certain advantageous embodiments, the suspension is a partial drawfrom an algal growth container, e.g., a draw taken 1, 2, or 3 times perday, or a draw taken once every 1, 2, 3, 4, 5, 6, or 7 days. Generally,the partial draw consists of approximately 10, 20, 30, 40, 50, 60, 70,80, or 90 percent of the culture volume from an algal growth containeror is in a range of 10 to 30, 30 to 50, 50 to 70, or 70 to 90 percent ofthe culture volume. Lysed and/or flocculated algal cells are separatedfrom water in the suspension to provide recovered water, and therecovered water is sterilized and returned to the algal growthcontainer.

In another embodiment, a system for processing algal cells in suspensionincludes a growth container in which algal cells are grown insuspension; a static mixer fluidly connected with the container throughwhich at least part of the suspension is passed, thereby lysing at leastsome of said cells; and electrolysis electrodes in contact with thesuspension, wherein an EMP is passed through the electrodes and throughsuspension between the electrodes.

In certain embodiments, the static mixer includes an injection portthrough which fluid may be entrained in the suspension; the static mixeralso includes anode and cathode electrodes electrically connected to anelectrical power source, e.g., as described herein.

In certain embodiments, the system also includes a biomass separator, alipid extractor, and/or a hydrogen collector.

Some embodiments include a modified static mixer. Such a modified staticmixer includes a body having a mixing throat through which liquid ispassed, an injection port whereby fluid materials may be entrained insaid liquid, and anode and cathode electrodes electrically separatedfrom each other such that when a voltage is applied across saidelectrodes, an electrical current will pass through said liquid.

While such a mixer may be configured in many ways, in certainembodiments, one of the electrodes is within the body, and the other ofthe electrodes is located at the outlet in the body; one of theelectrodes consists essentially of the body of the mixer, and the otherof the electrodes consists essentially of an outlet ring insulated fromthe body.

Utilization of algae in methods for producing large quantities of algaloil or algal biomass has faced a number of hurdles. In addition toachieving efficient growth, those hurdles include efficiently separatingalgae biomass from culture fluid and lysing of cells to enableseparation of oils or other products from cellular mass and debris. Theproblems are dramatically increased in large scale operations incontrast to laboratory scale processes. Indeed, many laboratory scaleprocesses are not applicable to large scale operations due to physicallimitations and/or cost limitations.

For example, in investigating these matters, no suggestion has beenfound for industrial scale application of EMP to the cell lysis oforganisms of the taxonomy group: Archeaplastida and particularly its subgroup micro-algae. Indeed, conventional methods focus mainly onelectrolysis of sludge (i.e., municipal and industrial waste) which islower in pH and therefore has a higher or positive Oxygen ReductionPotential (ORP) or Mv reading.

Electrochemically, as pH lowers, there is a dramatic increase in theconcentration of hydrogen ions and a decrease in negative hydroxyls orOH-ions (J. M. Chesworth, T. Stuchbury, J. R. Scaife, Introduction toAgricultural Biochemistry, pg. 12. 2.2) Conversely, the higher the pH,the lower the ORP. This correlation between high pH and negative Mvreadings led to the conclusion that a resident charge on the cell wallcan be transformed as energy to both facilitate cell lysing, but also toextract desired elements within the cell of benefit for the productionof energy, pharmaceuticals and food products. From recent advances inX-ray crystallography biology of single cell organism, in this casecyanobacteria or blue green algae, it was concluded that plant cellmembranes are like the two ends of a battery, they are positive on theinside and negative on the outside, and they are charged up when solarenergy excites electrons from hydrogen within the cell. The electronstravel up into the cell membrane via proteins that conduct them justlike wires releasing the energy a plant needs to stay alive and fromdata on the accumulation of tetraphenylphosphonium within Chlorellavulgaris cells, it can be estimated that these cells possess a membranepotential of −120 to −150 mV.

This negative potential is reflected in the observation of a vibrantcell colony's matrix pH level, where this measurement along with thecorrelate ORP (Mv reading) were taken to determine cell colony health.For example, a pH reading of 7 in an algae growth vessel correlate to anORP reading of (+/−) +200 Mv. When good cell health or log growth isattained; the pH of the matrix was noted to be pH 9.0; the corollary ORPreading was (+/−) −200 Mv. Therefore, it can be surmised that themeasure of a healthy algae cell colony can be determined by a negativeMy reading with each increase in one point of pH correlating to adecrease of roughly 200 Mv.

Most natural waters have pH values of between 5.0 and 8.5. As plantstake in CO2 for photosynthesis in aquatic ecosystems, pH values (andalkalinity) rise. Aquatic animals produce the opposite effect—as animalstake in 0₂ and give off CO₂, the pH (and acidity) is lowered. In steadystate, the algae matrix reading was 7.0 pH and as hypertonic conditionsare created through oxidation, the pH drops to below 7.0 and as low as5.0 with an analogous ORP reading of +200 to +400 Mv. When the cell walldoes not collapse, but just becomes flaccid (as opposed to turgid); itscontents are still encysted and the cell wall as represented in Donnan'slaw of equilibrium where the cell wall sets up an energy potentialwithin its two opposite charged cell walls to survive until an isotonicstate is regained. This is also referred to as the Gibbs-Donnanphenomenon. This is the state of equilibrium existing at a semipermeablemembrane when it separates two solutions containing electrolytes, theions of some of which are able to permeate the membrane and the othersnot; the distribution of the ions in the two solutions becomescomplicated so that an electrical potential develops between the twosides of the membrane and the two solutions have different osmoticpressures. This charge is extremely balanced and is why cells cansurvive extreme adverse conditions only to rejuvenate when properhypotonic conditions are present.

Live algal cells can be considered as an electrochemical fuel cell,where changing the polarity of the membrane from a live culture high pHand low ORP (150 Mv) to a low pH and high ORP (+200 Mv) results in thenet gain of 350 My and an attendant release of hydrogen into the matrix,provided the electrical potential of the cell is broken and the cellwall is not just deflated. Such hydrogen production is one of thebeneficial products obtainable from this invention.

By combining a number of approaches, it was discovered that a rapid,industrially scalable method of lysing and/or flocculating algal cellscan be provided. Such methods can be applied in methods for obtaininguseful products from algae, for example, extracting lipids, obtaininghydrogen gas, and/or obtaining algal cellular mass and debris, amongothers.

As a component to carry out such a process, the present methods can usea static mixer. Advantageous static mixers include but are not limitedto those described in Uematsu et al., U.S. Pat. No. 6,279,611, Mazzei,U.S. Pat. No. 6,730,214. Such mixers that assist in the generation oftransient cavitation and/or mass transfer of gas to liquid can be used.

It is surmised that by creating a rapid increase in ORP throughmanipulation or lowering the pH of the matrix, the electricaldifferential has the effect of abetting the electrolysis process in celllysing with the attendant benefit of the generation of excess hydrogenas a byproduct of the cell wall content release.

Experimental work demonstrates that cell lysing was realized rapidly andeconomically with this combination. The theory of why a combination ofcavitation, ultrasonics and pH modification works to lyse cells isempirical and the inventors are not intending to be bound by anyparticular explanation of the results.

The present process can advantageously include modification of ORP,usually through pH reduction. While such pH reduction (or other ORPmodification) can be accomplished using a variety of acids and bases, itcan also be accomplished using CO2. Oxidation/reduction reactionsinvolve an exchange of electrons between two atoms. The atom that losesan electron in the process is said to be “oxidized.” The one that gainsan electron is said to be “reduced.” In picking up that extra electron,it loses the electrical energy that makes it “hungry” for moreelectrons. Chemicals like chlorine, bromine, and ozone are alloxidizers.

ORP is typically measured by measuring electrical potential or voltagegenerated when a metal is placed in water in the presence of oxidizingand reducing agents. These voltages give us an indication of the abilityof the oxidizers in the water to keep it free from contaminants. Thus,an ORP probe is really a millivolt meter, measuring the voltage across acircuit formed by a reference electrode constructed of silver wire (ineffect, the negative pole of the circuit), and a measuring electrodeconstructed of a platinum band (the positive pole), with the fluid beingmeasured in between. The reference electrode, usually made of silver, issurrounded by salt (electrolyte) solution that produces another tinyvoltage. But the voltage produced by the reference electrode is constantand stable, so it forms a reference against which the voltage generatedby the platinum measuring electrode and the oxidizers in the water maybe compared. The difference in voltage between the two electrodes ismeasured.

Changing the pH of an aqueous solution can dramatically alter the ORPreading because of the effect of pH on the concentration of charged ionsin the water. Thus, in the apparatuses and methods described herein, thepH and thus the ORP can be modified by contacting the water with one ormore ORP or pH modifying agents. Advantageously, carbon dioxide gas canbe used to lower the pH; bringing the pH down will raise the millivoltreading.

CO₂ can be entrained in the liquid medium in the form of micro ornanobubbles, e.g., entrained as micro or nanobubbles using a staticmixer as described above. Entrainment of CO₂ gas in such a manner lowersthe pH, modifying the ORP, which can lead to the production ofadditional hydrogen gas which can be collected.

In addition, entrainment of CO₂ (or other gas) as micro or nanobubblescan contribute to cell lysis as indicated below. Cavitation effectsand/or ultrasonics can also be beneficially utilized to enhance celllysis and/or cellular mass and debris flocculation. While such effectscan be generated using an ultrasonic probe, they can also be generatedusing the cavitation effect of a static mixer with associatedmicrobubble entrainment. Thus, passing the algae-containing mediumthrough a static mixer with gas entrainment contributes to cell ruptureand can assist cellular mass and debris flocculation.

As applied in the present system, EMP has the effect of lysing cells.However, an added benefit is the generation of hydrogen gas, which canbe collected, e.g., for use as a fuel. The quantity of hydrogen can beenhanced by ORP modification.

For some applications, it may also be beneficial to apply a magneticfield. For example, such a field can be applied in or adjacent to astatic mixer. One way of accomplishing this is to locate strong magnetsaround the static mixer. In some cases, it may be beneficial to usealternating magnetic fields.

The present process can be configured to enhance the output of one ormore of a number of different products. For example, products can bealgal cellular mass and debris, lipids, selected proteins, carotenoids,and/or hydrogen gas.

In some applications, it may be desirable to generate cellular mass anddebris using the methods and apparatuses described herein. Such cellularmass and debris can be produced in conjunction with enhanced oroptimized production of one or more other products, or either withoutobtaining other products or without optimizing for obtaining otherproducts.

Advantageously, the process can be configured to produce substantialamounts of hydrogen gas.

In a typical embodiment, it is desirable to obtain lipids from thealgae, e.g., for use in biofuels and/or to provide algal omega-3 fattyacid containing oils (primarily eicosapentaenoic acid (20:5, n-3; EPA)and docosahexaenoic acid (22:6, n-3; DHA). For extracting such lipids,it is advantageous to lyse the cells, e.g., as described above. Releaseof lipids in such a manner allows a first separation to be carried outon the basis of different densities between the lipid-containingmaterial and the bulk water. If desired, the lipids can be furtherextracted using other lipid extraction methods.

In some embodiments, this invention utilizes a plurality of theprocesses mentioned to produce enhanced cellular mass and debrisseparation, cell lysis, hydrogen production, and/or lipid separation.For example, electrolysis can be combined with ORP modification.

Highly advantageously, a system is constructed to carry out the selectedsub-processes as part of the overall algae processing method. Onecomponent useful in such a system utilizes a modified static mixer whichhas an anode and cathode built into the device. In use, the modifiedstatic mixer subjects the slurry to EMP, while concurrently injectingCO₂ gas or other ORP modifying agent through a venturi into the algaeliquor as it flows through the device. The device can include a gasrecovery system on either end for the recovery of gases (e.g., hydrogen)generated by the electrolysis process.

Such a modified static mixer is schematically illustrated in FIG. 15.Biomass slurry 601 is allowed entry into the mixer chamber via an intakepipe. Once inside the entry chamber the slurry 601 flows through ananode 602 and cathode 603 circuits which is powered by a direct currentpower supply 654. The anode and cathode electrodes, 602 and 603, onlyallow electrical transfers when a conductive liquid medium is flowedbetween them. In the case of this static mixer, the biomass slurry 601is used to conduct the electrical transfer between the anode and cathodeelectrodes, 602 and 603. During electrical transfer, the biomass slurry601 is further exposed to the transfer and with a partial amount of thistransfer absorbed by the microorganism cells. Once electrical exposureoccurs their cellular wall structures begin to weaken. After flowingthrough the anode and cathode circuit chamber, a non-conductive gasket655 is used to isolate the two chambers apart as so to not allow andelectrical transfer to the venturi chamber 656. The now structurallyweaker cells can now be fractured by cellular/micron bubble collisioncaused by the venturi. To further increase efficiency of the substanceseparation process, a gas injection port 657 can be used to introducechemical enhancements for substance fracturing and recovery. Duringcellular wall fracturing, a release of intercellular gases such asoxygen and hydrogen or others having value can be captured as part ofthe substance recovery system. These gases are directed to vent forcapture at the end of the outlet 658 located at the static mixer exitport 659. Further exiting are the remains of the fractured biomass 629which is also directed for recovery at the exit point 658.

Thus, as indicated above, the system can advantageously be configuredand used with partial draws from the growth container or reactor, e.g.,a photo bioreactor. Also advantageously, the system can include and usea modified static mixer as described for extracting and flocculating(cellular mass and debris) from the matrix, capturing the generatedhydrogen or excess oxygen, separating the cellular mass and debris fromthe water and returning the water back to the reactor, preferably aftersterilization or filtration.

The method referred to herein as “Cascading Production”, makes use of apercentage draw of (culture) liquor from the growth tank on a scheduledbasis such as daily, every other day or weekly. The drawn (culture)liquor is then entrained through the electrolyzing mixing device and/orentrained through a mixer in conjunction with conventional electrolyzingmethod, such as an anode and cathode plate in the processing tank. Suchprocessing can include ORP manipulation.

Viewed in a general sense, the methods and apparatuses described hereininclude a series of fluid manipulations along a process flow with thespecific goal of extracting valuable by-products contained in algalcells. As described briefly above, as the algae is grown in tanks, e.g.,salt water tanks, of diverse configurations such as outdoor growthponds, open tanks, covered tanks, or photo bioreactors (PBR), a portionof the solution or liquor is drawn on a scheduled basis. This drawschedule is determined but not limited to the following observationstaken on a daily basis of density, pH and/or ORP. For example, it hasbeen noted that the pH of an outdoor pond is higher in the evening thanduring the morning, due to CO₂ absorption and the process referred to asrespiration which occurs at night. The difference can be as high as 3 pHpoints or 600 Mv. Therefore, one would draw a significant portion of thegrowth pond in the evening as the pH is now at 8.5-10 (early morningreadings would compare at (6.-7). In a reactor or PBR, the sameprinciple applies, but in this case one observes the log stages ofgrowth and draws up to 75% of the growth fluid (matrix) when the pHreaches 8.5-9. All these indicators use conventional measuring equipmentincorporated into a plant process computer controller, that wouldcontrol the extraction process and signal when it is time to harvest. Todetermine when it is time to harvest, several indicators in the growthvessel, such as PH, ORP, Mv, salinity, size of cells, etc., can beevaluated.

The remaining percentage of undrawn fluid is kept as an incubator forthe recycled water and used to start a new log phase of algae growth.The drawn liquor (also referred to herein as “culture”).

Microorganism algae are grown in a containment system and at the end ofan appropriate growth cycle are transferred into the substance recoveryprocess. The algae biomass are flowed through an optional micron bubblecavitation step, used to soften the outer cellular wall structure priorto other bio substance recovery processes.

After the cavitation step an optional heat process can be applied tochange the gravity specifies of the liquid feed stock water containingthe biomass. The heat option allows a faster transfer of particularsubstances released during the harvest process. After the biomass hasreached an appropriate heat range, it is then allowed to flow through anelectromagnetic pulse field, the EMP station where transiting biomasscells are exposed to the electromagnetic transfers resulting in thefracturing of the outer cellular wall structures.

Once flowed through the EMP process, the fractured biomass transitionsinto a gravity clarifier tank where heavier matter (cellular mass anddebris) sinks down through the water column as the lighter matter risesto the surface where it allows an easier harvest. The heavier sinkingmaterial (cellular mass and debris) gathers at the bottom of theclarifier tank where it can be easily harvested for other usefulsubstances. After substance separation and recovery, the remainder ofthe water column is sent through a water reclaiming process and afterprocessing is returned back into the growth system.

During this period of “cracking”, the static mixer can inject one ormore ORP modifiers, which can be or include pH modifiers such as CO₂.While CO₂ is preferred, alternative or additional pH or ORP modifierscan be used which accomplish the basic function of altering the pH valueand its corollary ORP value as represented in Mv. Any suitable staticmixer can be used; the methods, systems and apparatuses described hereinare not limited to any particular type of mixer or the associatedelectrolyzing method. Such a mixer can incorporate a cathode and anodeconnected to a voltage regulator, which preferably flips polarities soas to reduce scaling on the probes. The anode and cathode are powered bya DC energy source, such as a battery, generator, transformer orcombination thereof. The DC voltage can be set to variable outputs toadjust to algae mass in the cracking tank.

As the fluid is entrained through the Venturi mixer, it is thereforeadmixed with CO₂, subjected to EMP field as mentioned above, and throughthe continuous mixing, a plurality of micron bubbles are generated,creating a cavitated, or slurry of micron bubbles of both CO₂ and algamass. A combination of CO₂ entrainment, electrolysis, and mixing can beempirically selected, e.g., based on the desired separation of productsfrom the algae cells and/or flocculation of the mass to the surface ofthe water.

For example, in a recent test, CO₂ was applied to attain a drop from pH8.5 to 6.5 with a corresponding increase from −200 Mv to +250 Mv and thefluid was electrolyzed using a DC 6 Volts power supply and completeflocculation and cell lysing (as examined under a microscope) wasobtained within a period of 20 minutes. However, this combination andthese parameters are only exemplary, and can be examined to determineoptimum values. Desired results can be further correlated withprocessing variables, e.g., to establish protocols based on pH values,ORP reading, cell density and alga species. Upstream PH modification,prior to extraction, may help the emf extraction process.

When electrolysis is utilized, concurrent with the process of cracking(lysing) hydrogen gas (H+) is released at the cathode. This hydrogen canbe safely recovered and trapped in a tank through a hydrogen recoveryvalve, placed on the cathode end of an electrolyzing unit or at the endof the static mixer. If one alters the pH values by using a basechemical compound, e.g., potassium hydroxide, sodium hydroxide, calciumhydroxide or magnesium hydroxide, one would now create an excess of freeoxygen at the anode probe. In this instance, one would draw as above acertain portion of algae mass at a pH value of 8.5 and raise that valueto approximately pH 11 or roughly −250 Mv to −700 My and create a matrixhigh in negative hydroxyls or —OH. The dissociation of the free oxygenwould then be created as the matrix returned to 7.0 upon cell cracking.In this case, one would incorporate a safe recovery system for thisoxygen.

In this system, once the cellular mass and debris is cracked, dependingon the conditions, it may flocculate to the surface of the water or maysink. The cellular mass and debris is generally a composite of brokencell wall, lipid, carbohydrate and chlorophyll (A). In many cases,within a few hours, floc at the surface sinks to the bottom of the tank.While some of the lipid may remain on the surface, a significantfraction of the lipid (which may be most of the lipid) is stillassociated with chlorophyll and/or other cellular components and willsink with the rest of the cellular mass and debris.

The remainder of the water is now of about 7.0 pH, with a high CO2concentration. (only if pH was adjusted, otherwise the PH will be thatof the inbound slurry) This water (slurry is processed) and its crackedbiomass (cellular mass and debris) is now entrained or flowed to a watersterilizing tank after passing through a filtration unit, where a numberof systems can be used to separate out the organic mass from the water.These systems can, for example, be plane separators, filters, vortexseparators or any other method that performs the function of deliveringa separated mass. The separated cellular mass and debris is drawn to acellular mass and debris collection vessel and the water is sent on forsterilization in tank. After sterilization, the recovered water can beused to replenish tank.

In one embodiment, the system includes a modified Venturi mixer nozzle,e.g., as illustrated in FIG. 13. As previously indicated, the slurryinput pipe is insulated in the middle, or anywhere else along the lengthof pipe with a large rubber gasket or other electrically insulatingmaterial so as to separate the polarity of the anode and cathode. Thetwo ends of the tube can be electrified from source DC input or includeprobes within the tubes that have the purpose of conducting electricity.The modified Venturi introduces CO₂ gas or other admixture with thepurpose of altering pH and ORP through an inlet tube into a low pressurezone designed within the geometry of the tube; according to Bernoulli'sprinciple. At the exit of the venturi tube, a device can be installedfor the purpose of capturing the hydrogen created during the EMPprocess. One can add obstructions within the venturi tube to impact thefluids flow to increase turbulence and create a plurality ofmicron-bubbles.

Example 2 Quantification of Lipid Extraction and Identification ofOptimal EMP Extraction Parameters

In the experiments described below, quantification of lipid extractionusing an EMP apparatus as described herein and identification of optimalextraction parameters are described. The results described belowcorrespond to the data in FIG. 16.

Test 1:

In order to quantify lipid extraction from an EMP unit as describedherein, the following experiment was performed. A batch ofNannochloropsis oculata was processed through the 6-inch EMP unit toextract the lipids. The batch was gravity fed through the EMP unit at aflow rate of about 1 L/min A total of 20.8 L of algae culture wasprocessed. The processed stream was scooped off the top layer aftercollection for lipid analysis.

Control Batch Details:

Dry mass concentration: 433 mg/LLipid content: 5.5% of dry mass (23.86 mg/L)pH: 7.1Conductivity: 8.82 mS/cm

Extraction Process Details:

Extraction sample volume: 20.8 LFlow rate: 1 L/min

Voltage: 4.3 V

Electric current: 22 Amp

Results: The extraction sample was analyzed by the Folch method. Theextracted lipid weighed 0.4481 g. The amount of lipid originally presentin the 20.8 L of algae batch before processing was 0.4965 g. Thiscorresponds to an extraction efficiency of 90.2% through the EMP unit.

Test 2:

In order to quantify lipid extraction from an EMP unit as describedherein, the following experiment was performed. A batch ofNannochloropsis oculata was processed through the 6-inch EMP unit toextract the lipids. The batch was gravity fed through the EMP unit at aflow rate of about 1 L/min A total of 9.2 L of algae culture wasprocessed. The processed stream was collected in a lipid collectionapparatus that was designed to have tapered long neck to collect thelipid layer floating at the top.

Control Batch Details:

Dry mass concentration: 207 mg/LLipid content: 13% of dry mass (26.91 mg/L)pH: 6.8Conductivity: 9.31 mS/cm

Extraction Process Details:

Extraction sample volume: 9.2 LFlow rate: 1 L/min

Voltage: 3.4 V

Electric current: 20 Amp

Results: The extraction sample was analyzed by the Folch method. Theextracted lipid weighed 0.2184 g. The amount of lipid originally presentin the 9.2 L of algae batch before processing was 0.2477 g. Thiscorresponds to an extraction efficiency of 88.2% through the EMP unit.

Test 3:

In order to quantify lipid extraction from an EMP unit as describedherein, the following experiment was performed. A batch ofNannochloropsis oculata was processed through the 6-inch EMP unit toextract the lipids. The batch was gravity fed through the EMP unit at aflow rate of about 1 L/min A total of 11 L of algae culture wasprocessed. The processed stream was scooped off the top layer aftercollection for lipid analysis.

Control Batch Details:

Dry mass concentration: 207 mg/LLipid content: 13% of dry mass (26.91 mg/L)pH: 6.8Conductivity: 9.31 mS/cm

Extraction Process Details:

Extraction sample volume: 11 LFlow rate: 1 L/min

Voltage: 3.4 V

Electric current: 20 Amp

Results: The extraction efficiency was 95.25% through the 6-inch EMPunit for the tested algae batch.

Test 4:

In order to quantify lipid extraction from an EMP unit as describedherein, the following experiment was performed. A batch ofNannochloropsis oculata was processed through the 6-inch EMP unit toextract the lipids. The batch flow rate was regulated using a flowmeterand a pump. 2 liters of algae culture was processed. The processedstream was collected in a 2 liter volumetric flask, and the top lipidlayer was recovered for analysis.

Control Batch Details:

Dry mass concentration: 410 mg/LLipid content: 8.2% of dry mass (33.62 mg/L)pH: 7.1Conductivity: 8.99 mS/cm

Extraction Process Details:

Extraction sample volume: 2.01 LFlow rate: 1.5 L/min

Voltage: 12.4 V

Electric current: 18 Amp

Results: The extraction efficiency was 90.7% through the 6-inch EMP unitfor the tested algae batch.

Test 5:

In order to quantify lipid extraction from an EMP unit as describedherein, the following experiment was performed. A batch ofNannochloropsis oculata was processed through the 12-inch EMP unit toextract the lipids. The batch flow rate was regulated using a flowmeterand a pump. 1.87 liters of algae culture was processed. The processedstream was collected in a 2 liter volumetric flask, and the top lipidlayer was recovered for analysis.

Control Batch Details:

Dry mass concentration: 800 mg/LLipid content: 19.9% of dry mass (159.2 mg/L)pH: 7.6Conductivity: 8.15 mS/cm

Extraction Process Details:

Extraction sample volume: 1.87 LFlow rate: 0.2 gal/min (0.756 L/min)

Voltage: 4.8 V

Electric current: 20.2 Amp

Results: The extraction efficiency was 12.2% through the 12-inch EMPunit for the tested algae batch.

Test 6:

In order to quantify lipid extraction from an EMP unit as describedherein, the following experiment was performed. A batch ofNannochloropsis oculata was processed through the 12-inch EMP unit toextract the lipids. The batch flow rate was regulated using a flowmeterand a pump. 1.87 liters of algae culture was processed. The processedstream was collected in a 2 liter volumetric flask, and the top lipidlayer was recovered for analysis.

Control Batch Details:

Dry mass concentration: 500 mg/LLipid content: 16.15% of dry mass (80.75 mg/L)pH: 7.5Conductivity: 8.18 mS/cm

Extraction Process Details:

Extraction sample volume: 1.87 LFlow rate: 1.13 L/min

Voltage: 4.7 V

Electric current: 20 Amp

Results: The extraction efficiency was 51.5% through the 12-inch EMPunit for the tested algae batch.

Test 7:

In order to identify the optimal EMP extraction parameters for a givenalgae batch, the EMP was tested in a matrix of wide range of parameters.A batch of Nannochloropsis oculata was processed through the 6-inch EMPunit to extract the lipids. The batch flow rate was regulated using aflowmeter and a pump. Individual samples that comprised the matrix oftesting were collected in small 116 ml bottles. The cellular mass anddebris at the bottom and the water were syringed out leaving only thetop lipid layer in the extraction sample bottle.

Control Batch Details:

Dry mass concentration: 210 mg/LLipid content: 24% of dry mass (50 mg/L)pH: 7.8Conductivity: 7.89 mS/cm

Extraction Results:

Extraction sample volume: 116 ml

The amount of lipid originally present in the 116 ml algae sample beforeprocessing: 5.8 mg

The extraction sample was analyzed by the Folch method. The relevantparameters comprising the matrix of testing conditions and theextraction efficiency are tabulated in Table 1.

TABLE 1 Extraction efficiency at different flow rates and currentstrengths Current Flow rate 5 Amp 10 Amp 15 Amp 20 Amp 0.25 gal/minSample # 2 Sample # 5 Sample # 8 Sample # 10 (0.95 L/min) Voltage: 11.5V Voltage: 11.5 V Voltage: 11.5 V Voltage: 11.5 V Lipid extracted: Lipidextracted: Lipid extracted: Lipid extracted: 4.0 mg 4.2 mg 5.6 mg 5.2 mgEfficiency: 69% Efficiency: 72% Efficiency: 97% Efficiency: 90% 0.38gal/min Sample # 14 Sample # 17 Sample # 20 Sample # 23 (1.44 L/min)Voltage: 11.5 V Voltage: 11.5 V Voltage: 11.5 V Voltage: 11.5 V Lipidextracted: Lipid extracted: Lipid extracted: Lipid extracted: 3.0 mg 4.5mg 4.1 mg 4.5 mg Efficiency: 52% Efficiency: 78% Efficiency: 71%Efficiency: 78% 0.5 gal/min Sample # 26 Sample # 29 Sample # 32 Sample #35 (1.89 L/min) Voltage: 11.5 V Voltage: 11.5 V Voltage: 11.5 V Voltage:11.5 V Lipid extracted: Lipid extracted: Lipid extracted: Lipidextracted: 3.3 mg 3.2 mg 3.0 mg 2.6 mg Efficiency: 57% Efficiency: 55%Efficiency: 52% Efficiency: 45%

Inference: The most optimal conditions for lipid extraction for thisbatch of algae look to be 0.25 gal/min and 15 Amp. The efficiencydecreases gradually around this set of conditions in the tested matrix.At higher currents at 0.25 gal/min, the energy input is probably toohigh to the detriment of algae causing them to destruct. At lowercurrents at 0.25 gal/min, and at lower flow rates, the energy input istoo less to fully extract the lipids from algae.

Test 8:

In order to quantify lipid extraction from an EMP unit as describedherein, the following experiment was performed. A batch ofNannochloropsis oculata was processed through the 6-inch EMP unit toextract the lipids. The batch flow rate was regulated using a flowmeterand a pump. Samples were collected either in 116 ml bottles or 400 mlbottles. The cellular mass and debris at the bottom and the water weresyringed out leaving only the top lipid layer in the extraction samplebottles.

Control Batch Details:

Dry mass concentration: 320 mg/LLipid content: 18% of dry mass (57.6 mg/L)pH: 7.3Conductivity: 7.93 mS/cm

Extraction Process Details:

Flow rate: 0.95 L/min

Voltage: 5.3 V Current: 20 A Results:

Extraction sample 1:

Volume: 412 ml

Extraction efficiency: 83.31%Extraction sample 2:

Volume: 116 ml

Extraction efficiency: 80.69%Extraction sample 3:

Volume: 116 ml

Extraction efficiency: 95.64%

Test 9:

In order to identify the optimal EMP extraction parameters for a givenalgae batch, the EMP apparatus as described herein was tested in fourdifferent sets of conditions. 20 liters of a Nannochloropsis oculatabatch from the grow room was processed through the 6-inch EMP unit. Thebatch flow rate was regulated using a flowmeter and a pump.

Control Sample Details (Sample #1130-0):

Dry mass concentration: 320 mg/LLipid content: 11% of dry mass (35 mg/L)pH: 7.5Conductivity: 8.15 mS/cm

The algae batch was processed under various flow rate and energy inputconditions as listed below:

Sample 1130-3: Flow rate=0.25 gal/min, Voltage=3.7 V, Current=15 AmpSample 1130-4: Flow rate=0.25 gal/min, Voltage=4.0 V, Current=20 AmpSample 1130-8,9: Flow rate=0.38 gal/min, Voltage=4.0 V, Current=20 AmpSample 1130-12: Flow rate=0.38 gal/min, Voltage=3.7 V, Current=15 Amp

Samples were collected in 400 ml bottles. The cellular mass and debrisat the bottom and the water were syringed out leaving only the top lipidlayer in the extraction sample bottles. The samples were analyzed byCSULB-IIRMES using the Folch Method.

Results: The most optimal conditions for lipid extraction for this batchof algae look to be 0.38 gal/min; 3.7 V; 15 Amp.

TABLE 2 Extraction Lipid Content Lipid Sample Before ExtractionExtracted Extraction Sample # Volume (L) (mg/L) (mg/L) Efficiency 1130-30.38 35 25.3 72% 1130-4 0.38 35 27.9 80%  1130-8, 9 0.38 35 26.8 77% 1130-12 0.38 35 32.6 93%

Test 10:

The new Pipe EMP (i.e., pulsed emf) equipment along with MX cavitationand heat was tested and compared with previous tests. A batch ofNannochloropsis oculata was processed through the Pipe single stepextraction (herein “SSE”) system. The components of the Pipe SSE systemare the pipe EMP unit, a heat strip system around the pipe EMP unit, andan MX cavitation unit. The MX cavitation unit precedes the pipe EMPunit. The MX cavitation unit and the heating system around the EMP unitcould be used optionally. The cavitation was done for 1 minute. Thebatch flow rate was regulated using a flowmeter and a pump. Samples werecollected in 120 ml bottles. The cellular mass and debris at the bottomand the water were syringed out leaving only the top lipid layer in theextraction sample bottles.

Control Batch Details:

Dry mass concentration: 280 mg/LLipid content: 21% of dry masspH: 7.7Conductivity: 7.42 mS/cm

Extraction Results and Observations:

Extraction sample volume: 120 ml

TABLE 3 Extraction results and observations of the Pipe SSE testing thatincluded both MX cavitation and heating Current Flow rate gal/min) (Amp)0.25 0.50 1.00 2.00 5 Voltage = 2.1 V Voltage = 2.1 V cellular mass andAll cellular mass debris sank after 60 min and debris floated 10 Voltage= 3.1 V cellular mass and debris sank after 25 15 Voltage = 2.6 VVoltage 2.6 V Voltage = 2.6 V Voltage = 2.6 V cellular mass and Allcellular mass All cellular mass All cellular mass debris sank instantlyand debris floated and debris floated and debris floated ExtractionEfficiency = 66% Extraction Efficiency = 65% 20 Voltage = 3.8 V Voltage= 3.8 V cellular mass and cellular mass and debris debris sank instantlysank slowly (1 day) Note: Rate of heating was the same for differentflow rates. This means that at 0.50 gal/min, cellular mass and debrisreceived less heat than that at 0.25 gal/min

The following table (Table 4) shows the extraction results andobservations of the Pipe EMP testing that included only of MX cavitationand heating or neither. This can be used for comparison with the similartesting conditions in the table above.

TABLE 4 Extraction Results 0.50 gal/min; 15 Amp 1.00 gal/min; 15 Amp NoMX/No Voltage = 3.5 V Voltage = 3.5 V Heat cellular mass and cellularmass and debris was suspended debris was suspended Extraction Efficiency= 95% No MX/Heat Voltage = 2.5 V Voltage = 2.5 V All cellular mass andAll cellular mass debris floated and debris floated ExtractionEfficiency = 107% MX/No Voltage = 3.6 V Voltage = 3.6 V Heat cellularmass and cellular mass and debris was suspended debris was suspendedExtraction Efficiency = 50%

It looked like heat resulted in enhanced electrolysis that resulted inthe cellular mass and debris to flocculate better. When the heat washigh (as in @ 0.25 gal/min), all the flocculated cellular mass anddebris sunk leaving a clear thin lipid layer at the top. The sinking wasprobably because the density of heated water is markedly lower than thatof cellular mass and debris. When the heat is low (as in @ 0.50gal/min), all the flocculated cellular mass and debris remained at thetop stuck to the lipid. This is probably because the differentialdensities of water and cellular mass and debris is not big enough tocause instant sinking of cellular mass and debris, but the applied heatwas still enough to flocculate the cellular mass and debris. Either way,it was seen that when there was heat the cellular mass and debrisflocculated either at the top or at the bottom, but when there was noheat they remained suspended as seen normally with the previous 6-inchand 12-inch EMP units without heat.

Another strong possibility is that when the cellular mass and debrisflocculates and sinks to the bottom with the application of heat, someof the extracted lipid that was stuck to the cellular mass and debriscould be carried along with the cellular mass and debris to the bottom.As a result, the extraction efficiency as analyzed from the lipid at thetop clear layer could be lower. Conversely, when the cellular mass anddebris flocculated and floated at the top, even if all of the lipidsinside the algae cells may not have been extracted, the non-extractedlipids may still remain at the top along with the extracted lipids.

Another observation was the effect of current in sinking the cellularmass and debris when heat was applied. In the first table, in the columncorresponding to 0.25 gal/min, the speed at which the cellular mass anddebris sank was directly proportional to the amount of electric currentsupplied. Even at the flow rate 0.50 gal/min, where all the cellularmass and debris floated because of lower heat, the cellular mass anddebris corresponding to the sample with 20 Amperes of electric currentsank after 1 day, whereas the cellular mass and debris corresponding tothe samples with lower current continued to float after 1 day.

Test 11:

In order to obtain lipid extraction at the highest efficiency possiblefor a given batch of algae, an EMP apparatus as described herein wastested in different sets of conditions. A batch of Nannochloropsisoculata was processed through the 6-inch EMP unit to extract the lipids.The batch flow rate was regulated using a flowmeter and a pump. Sampleswere collected in 1 liter bottles. The cellular mass and debris at thebottom and the water were syringed out leaving only the top lipid layerin the extraction sample bottles.

Control Sample Details (Sample #20100104-10):

Dry mass concentration: 285 mg/LLipid content: 6.67% of dry mass (19 mg/L)pH: 8.4Conductivity: 7.99 mS/cm

Extraction Results:

Extraction sample volume: 1 LThe amount of lipid originally present in the 1 L algae sample beforeprocessing: 19 mg

The samples were analyzed by CSULB-IIRMES using the Folch Method. Therelevant parameters of different testing conditions and the extractionefficiencies are tabulated in following table.

TABLE 5 Parameters of Testing Conditions and Extraction EfficienciesFlow rate: 0.25 gal/min Flow rate: 0.50 gal/min (0.945 L/min) (1.89L/min) Sample # 20100104-11 Sample # 20100104-16 Current: 12 AmpCurrent: 20 Amp Voltage: 3.5 V Voltage: 3.9 V Extraction efficiency: 45%Extraction efficiency: 67% Sample # 20100104-12 Sample # 20100104-17Current: 14 Amp Current: 18 Amp Voltage: 3.7 V Voltage: 3.8 V Extractionefficiency: 31% Extraction efficiency: 96% Sample # 20100104-13 Sample #20100104-18 Current: 15 Amp Current: 15 Amp Voltage: 3.7 V Voltage: 3.7V Extraction efficiency: 39% Extraction efficiency: 69% Sample #20100104-14 Current: 20 Amp Voltage: 4.0 V Extraction efficiency: 41%Sample # 20100104-15 Current: 19 Amp Voltage: 3.9 V Extractionefficiency: 98%

The highest extraction efficiencies 98% and 96% were obtained at 0.25gal/min; 19 Amp; 3.9 V and at 0.50 gal/min; 18 Amp; 3.8 V for the testedalgae batch.

Tests 12 and 13:

The effect of overnight storing in darkness and cold on lipid extractionefficiency was examined. Samples from the same algae batch were testedin Test 12 and were tested on the following day in Test 13. The samealgae batch tested in Test 12 was tested on the following day (the sametests were run on the same original algae culture; one test occurred onthe day the live sample was drawn from the growth tank, i.e., real-time,and the 2^(nd) day the remainder of the sample was tested after itrested overnight). A batch of Nannochloropsis oculata was processedthrough the Pipe SSE system. The components of the Pipe SSE system arethe pipe EMP unit, a heat strip system around the pipe EMP unit, and anMX cavitation unit. The MX cavitation unit precedes the pipe EMP unit.The MX cavitation unit and the heating system around the EMP unit couldbe used optionally. The cavitation was done for 1 minute. The batch flowrate was regulated using a flowmeter and a pump. Samples were collectedin 120 ml bottles. The cellular mass and debris at the bottom and thewater were syringed out leaving only the top lipid layer in theextraction sample bottles.

TABLE 6 The control sample details pertaining to the first day and thesecond day after storage. Control Sample- Test 12 Control Sample-Following day Dry mass concentration: 255 mg/L Dry mass concentration:270 mg/L Lipid content: 15.13% of dry mass Lipid content: 14.72% of drymass (38.57 mg/L) (39.74 mg/L) pH: 7.4 pH: 7.4 Conductivity: 7.64 mS/cmConductivity: 7.74 mS/cm

Extraction Results:

Extraction sample volume: 120 ml

TABLE 7 Relevant parameters of the testing conditions and the extractionefficiencies Test 12 The Following Day (Lipid content of algae (Lipidcontent of algae in 120 ml: 4.63 mg) in 120 ml: 4.77 mg) Sample # 1, 2Flow rate: 0.50 gal/min Voltage: 3.8 Current: 15 A MX, No HeatExtraction Efficiency: 16% Sample # 3, 4 Flow rate: 0.25 gal/minVoltage: 4.1 Current: 19 A No MX, Heat Extraction Efficiency: 19% Sample# 5, 6 Sample # 25, 26 Flow rate: 0.50 gal/min Flow rate: 0.50 gal/minVoltage: 3.8 Voltage: 3.8 Current: 15 A Current: 15 A No MX, Heat No MX,Heat Extraction Efficiency: 23% Extraction Efficiency: 20% Sample # 7, 8Sample # 27, 28 Flow rate: 0.50 gal/min Flow rate: 0.50 gal/min Voltage:3.8 Voltage: 3.8 Current: 15 A Current: 15 A No MX, No Heat No MX, NoHeat Extraction Efficiency: 45% Extraction Efficiency: 25% Sample # 11,12 Sample # 19, 20 Flow rate: 1.00 gal/min Flow rate: 1.00 gal/minVoltage: 3.7 Voltage: 3.8 Current: 12 A Current: 12 A MX, Heat MX, HeatExtraction Efficiency: 21% Extraction Efficiency: 23% Sample # 13, 14Sample # 21, 22 Flow rate: 0.50 gal/min Flow rate: 0.50 gal/min Voltage:3.8 Voltage: 3.8 Current: 15 A Current: 15 A MX, Heat MX, HeatExtraction Efficiency: 24% Extraction Efficiency: 24% Sample # 15, 16Flow rate: 0.25 gal/min Voltage: 3.8 Current: 15 A MX, Heat ExtractionEfficiency: 22%

The extraction efficiencies are in general lower than the earlier PipeSSE experiments. This is probably because the extraction samples wereleft to sit too long before recovering the top lipid layer. Usuallythere is some cellular mass and debris that is found in the top lipidlayer, but all of it had sunk as a result of letting the samples sit fortoo long, and along with it some of the lipid could have sunk as well.Comparing the extraction efficiencies observed on the first day and thesecond day, there does not seem to be any improvement in extraction dueto the overnight storage in darkness and cold.

Example 3 Use of Cavitation and EMP to Harvest Carbohydrates andProteins

FIG. 14 shows results from a test procedure for harvesting carbohydratesand proteins from algae. The test procedure was performed as follows.The algae slurry was first processed through the EMP unit at roomtemperature. The EMP processed slurry was collected in a storage tank.It was then cavitated through the MX unit. The cavitated slurry was thenallowed to sit for a few minutes. A thick mass of algae cellular massand debris raised to the top and remained floated. The floating cellularmass and debris was collected off the top for analysis.

The algae samples collected through the Inverse SSE process was analyzedby Anresco Laboratories, San Francisco. The samples were analyzed forlipid, protein and carbohydrate content of the algae. The analysis byAnresco Laboratories gave the total mass of protein, lipid orcarbohydrate in a given sample (say ‘x’ mg).

The dry mass concentration of the algae batch processed (say ‘d1’ mg/L)was measured before the Inverse SSE process. The volume of the algaebatch collected in the storage tank from where the final floatingcellular mass and debris was collected off the top was also known (say‘V’ L). The dry mass concentration of the remnant solution after thecollection of floating cellular mass and debris off the top was alsomeasured (say ‘d2’ mg/L). From these the mass of algae cellular mass anddebris (say ‘M’ mg) collected off the top of the storage tank wascalculated as follows:

M=(d1−d2)×V

Then, the individual composition of protein, for example, was calculatedas follows:

Protein composition=x/M mg of protein/mg of algae dry mass.

For this experiment, three small samples were taken from the sample jar(it was observed that the algae collected off the top from the processwas sticky, agglomerated and floating on water). Based on the dry massmeasurements and the volume of algae slurry processed, the amount ofbiomass collected off the top through the Inverse SSE process was 600mg. The protein quantity alone as analyzed by Anresco Laboratoriesamounts to 1400 mg. As the amount of protein should not be higher thanthe amount of biomass, the amounts measured could be due to increasedprotein numbers that resulted from sampling methods, e.g., there mighthave been more algae in the three drawn samples than there might be ifthey were uniformly mixed. Nonetheless, these results demonstrate thatthe apparatuses and methods described herein can be used to harvestprotein as well as fat from algae cells (see Table 8 below).

TABLE 8 Results from three samples of Algae marked 0413: 1-3 Sample IDAnalysis Findings #1 Protein (NX6.25) 0.70% #2 Fat #3 Fat

OTHER EMBODIMENTS

One skilled in the art would readily appreciate that the presentinvention is well adapted to obtain the ends and advantages mentioned,as well as those inherent therein. The methods, systems, and apparatusesdescribed herein as presently representative of preferred embodimentsare exemplary and are not intended as limitations on the scope of theinvention. Changes therein and other uses will occur to those skilled inthe art, which are encompassed within the spirit of the invention andare defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention. Forexample, variations can be made to the configuration of the tanks,materials utilized, ORP modifying agents, and algal species grown. Thus,such additional embodiments are within the scope of the presentinvention and the following claims.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions, any equivalents of thefeatures shown and described or portions thereof are excluded, but it isrecognized that various modifications are possible within the scope ofthe invention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by exemplaryembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

Also, unless indicated to the contrary, where various numerical valuesor value range endpoints are provided for embodiments, additionalembodiments are described by taking any two different values as theendpoints of a range or by taking two different range endpoints fromspecified ranges as the endpoints of an additional range. Such rangesare also within the scope of the described invention. Further,specification of a numerical range including values greater than oneincludes specific description of each integer value within that range.

Thus, additional embodiments are within the scope of the invention andwithin the following claims.

1-10. (canceled)
 11. A method for extracting non-polar lipids frommicroalgae in a flowing aqueous slurry, comprising: providing an aqueousslurry comprising microalgae; providing a lipid extraction apparatushaving a body including a channel that defines a fluid flow path,wherein a cathode and an anode form at least a portion of the channelthat defines the fluid flow path, the cathode and the anode being spacedapart to form a gap with a distance in a range from one of 0.5 mm to 200mm, or 1 mm to 50 mm within the channel; flowing the aqueous slurrythrough the channel and applying an electromotive force across the gapthat compromises the microalgae cells and releases a lipid fraction; andrecovering at least a portion of the nonpolar lipid fraction. 12-19.(canceled)
 20. The method of claim 11, wherein flowing the aqueousslurry through the channel and applying an electromotive force acrossthe gap that compromises the microalgae cells releases a lipid fractionhaving greater than 80 wt % non-polar lipids and less than 20 wt % polarlipids, having greater than 90% wt % non-polar lipids and less than 10wt % polar lipids, having greater than 95% wt % non-polar lipids andless than 5 wt % polar lipids, or having greater than 99 wt % non-polarlipids and less than 1 wt % polar lipids.
 21. The method of claim 11,wherein the aqueous slurry is caused to flow through the gap at a ratein a range from at least 0.1 ml per second per ml of gap volume to atleast 1.0 ml per second per ml of gap volume.
 22. The method of claim11, wherein the volume of the fluid flow path within the gap is in arange selected from a list consisting of at least 50 ml, at least 200ml, or at least 500 ml, or at least 1 liter.
 23. The method of claim 11,wherein at least 70 wt % of microorganism within the aqueous slurry aremicroalgae.
 24. The method of claim 11, wherein the electromotive forceis pulsed at a frequency selected from a list consisting of at least 500Hz, at least 1 kHz, at least 2 kHz, and at least 30 kHz.
 25. The methodof claim 11, wherein the amperage used to create the electromotive forceis selected from a list consisting of at least 1 amp, at least 5 amps,at least 10 amps, at least 50 amps, and at least 100 amps.
 26. Themethod of claim 11, wherein the voltage is in a range selected from alist consisting of at least 1V, at least 10 V, at least 100 V, at least1 kV, and at least 20 kV.
 27. The method of claim 11, wherein at least90 wt % of microorganism within the aqueous slurry are microalgae. 28.The method of claim 11, wherein the temperature of the aqueous slurryduring extraction is selected from a list consisting of at least 40° F.,at least 65° F., at least 80° F., at least 100° F., and at least 120° F.29. The method of claim 11, wherein the pH of aqueous slurry is in arange selected from a list consisting of from 6.6-9.0, 6.8-8.6, and7.0-8.5.
 30. The method of claim 11, wherein the pH of aqueous slurry isalkaline.
 31. A lipid extraction apparatus for extracting non-polarlipids from microalgae, comprising: a body including a channel thatdefines a fluid flow path from a first opening to a second opening, thefirst opening providing an inlet for an aqueous algae slurry and thesecond opening providing an outlet for the aqueous algae slurry; and acathode, an anode, and an insulator forming at least a portion of thechannel that defines the fluid flow path, the cathode and the anodebeing spaced apart to form a gap with a distance in a range selectedfrom 0.5 mm to 200 mm.
 32. An apparatus as in claim 31, wherein flowingthe aqueous slurry through the channel and applying an electromotiveforce across the gap that compromises the microalgae cells releases alipid fraction having greater than 80 wt % non-polar lipids and lessthan 20 wt % polar lipids, having greater than 95% wt % non-polar lipidsand less than 5 wt % polar lipids, having greater than 90% wt %non-polar lipids and less than 10 wt % polar lipids, or having greaterthan 99 wt % non-polar lipids and less than 1 wt % polar lipids.
 33. Anapparatus as in claim 31, wherein the volume of the fluid flow pathwithin the gap is at least 200 ml.
 34. An apparatus as in claim 31,wherein a surface area of the channel formed by the cathode and theanode is in a range from at least 500 cm² to at least 2000 cm².
 35. Anapparatus as in claim 31, wherein the body comprises a first conductivetube within a second conductive tube and the insulator providesseparation between the first and second conductive tubes, the channelbeing formed from spacing between the first and second conductive tubes.36. An apparatus as in claim 31, wherein the gap volume is selected fromone of at least 50 ml, at least 200 ml or at least 500 ml, or at least 1liter.
 37. An apparatus as in claim 31, wherein the surface area of theanode and cathode exposed to the fluid flow is selected from one of atleast 500 cm², at least 1000 cm² or at least 2000 cm².
 38. An apparatusas in claim 31, further comprising a power supply configured to supplyamperage selected from a list consisting of at least 1 amp, at least 5amps, at least 10 amps, at least 50 amps, and at least 100 amps.
 39. Anapparatus as in claim 31, further comprising: a computer controlledlipid extraction apparatus that utilizes HPLC data to select theparameters that minimize polar lipids in the released lipid from,wherein the parameters are selected from a list consisting of flow rate,amperage, voltage and gap distance.
 40. An apparatus as in claim 31,wherein the flow rate through the gap volume is selected from a listconsisting of 0.1 ml/second per ml of gap volume, at least 0.5 ml/secondper ml of gap volume, at least 1.0 ml/second per ml of gap volume and atleast 1.5 ml/second per ml of gap volume.
 41. An apparatus as in claim31, wherein the voltage is selected from a list consisting of at least1V, at least 10 V, at least 100 V, at least 1 kV, and at least 20 kV.42. An apparatus as in claim 31, wherein the frequency of the emf pulsesis at least 500 Hz, 1 kHz, at least 2 kHz, and at least 30 kHz.
 43. Anapparatus as in claim 31, wherein the temperature of the aqueous slurryduring extraction is selected from a list consisting of at least 40° F.,at least 65° F., at least 80° F., at least 100° F., and at least 120° F.44. An apparatus as in claim 31, wherein the pH of aqueous slurry is ina range selected from a list consisting of from 6.6-9.0, 6.8-8.6, and7.0-8.5.
 45. An apparatus as in claim 31, wherein the pH of aqueousslurry is alkaline.